Dual C–Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C–Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (−1.8 ± 0.2 and −19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (−1.22 ± 0.08 and −1.2 ± 0.5‰), leading to distinctly different trends (ΛC–Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C–Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC–Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.


SMM3. Activity assays with cell suspensions of Dehalogenimonas
To test reductive dehalogenase enzyme activity, specific assays were set-up inside an anoxic glovebox. Anoxic 10 mL glass vials were used, containing 2 mL of an assay buffer  Further information is available in a previous study. 4 Briefly, aqueous phase detection limits were 0.021, 0.001 and 0.0004 µmol/bottle (60, 1.1 and 0.024 µg/L), respectively.
The GC-FID response to a headspace sample (0.5 mL) was calibrated to give the total mass of the compound in that bottle, 6 which was then converted to an aqueous-phase concentration using Henry's law constants. 7 The stoichiometry of 1,2-DBA biodegradation accounted for the amounts that carried over from the enrichment culture, i.e., which are approximately 1.0 ± 0.2 µmol/bottle (62 ± 10 µg/L) ethene and 0.005 ± 0.002 µmol/bottle (6.3 ± 1.9 µg/L) VB.

S6
A Dehalogenimonas-containing culture transforming 1,2-DCP (50 µM) to propene was maintained for more than five years in the UAB laboratory as described elsewhere. 8

SMM7. Calculation of apparent kinetic isotope effects (AKIEs)
Intrinsic KIEs are position specific whereas εbulk values are calculated from compoundaverage isotope data (eq. 2 in the main text). Therefore, observable εbulk values have to be converted into AKIEs in order to obtain information about the underlying reaction mechanisms. 10 For the calculation and interpretation of AKIEs a hypothesis about the reaction mechanism, or assumed reaction mechanism, is necessary. The effects of nonreacting positions within the molecule, as well as of intramolecular competition, are then taken into account using eq. S1 and S2, respectively, 10 where εrp is the isotopic fractionation at the reactive position, "n" is the number of atoms of the element considered, "x" is the number of reactive sites and "z" the number of identical reactive sites undergoing intramolecular competition. These equations assume the absence of secondary isotope effects. For carbon, secondary isotope effects are usually insignificant. 10 In symmetric molecules such as 1,2-DBA, all atoms are in equivalent reactive positions (n = x), and therefore, εrp is directly obtained from the slopes of the Rayleigh plots (Fig. 2). If the two C-Br bonds are broken in sequence (e.g., SN2-reaction), assuming that the first bond cleavage is the rate-determining step, then z = 2 in eq. S2 as both C-Br bonds compete for reaction.

Dehalococcoides.
In order to investigate the biodegradation products of 1,2-DBA in the experiments with Dehalococcoides, data collected by GC-FID measurement on single bottles that continued to be incubated (headspace monitoring) are shown in Figure S3. where "k" is the first order rate constant (h -1 ), "A" is the frequency factor (h -1 ), "R" is the gas constant (8.314 × 10 -3 kJ mol -1 K -1 ), "Ea" is the activation energy (kJ mol -1 ) and "T" is the absolute temperature (K). According to the "Ea" (112 kJ mol -1 ) and "A" (8.7 × 10 14 h -1 ) values determined by Groos et al. 11 the transformation rate is estimated to be 1.53 × 10 -5 h -1 (i.e., half-live of around 5 years). This rate was used to simulate the remaining amount of 1,2-DBA and the formation of VB considering hydrolysis/dehydrohalogenation as the sole reaction mechanism (Fig. S3). Groos et al. 11 (and references herein) indicated that 1,2-DBA dehydrohalogenation to VB accounted for less than 5% of 1,2-DBA degradation. Accordingly, in the present study the formation of VB was simulated assuming that it represents a 5% of the total 1,2-DBA transformed.
Using the parameters indicated above, a very low degradation rate of 1,2-DBA by